Detection of hydrogen peroxide

ABSTRACT

The present invention provides compounds useful for detection of hydrogen peroxide and methods of using same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/587,387, filed Aug. 16, 2012, now U.S. Pat. No. 8,785,652, whichclaims the benefit of U.S. Provisional Application No. 61/524,066, filedAug. 16, 2011, which are incorporated by reference herein.

FIELD OF THE INVENTION

The present invention provides an assay for detection of hydrogenperoxide.

BACKGROUND OF THE INVENTION

To remain healthy, cells, in particular mammalian cells, need tomaintain a balance between oxidizing and reducing conditions, sometimereferred to as redox state/potential. Reactive oxygen species (ROS),including peroxides, are implicated in cellular activity and metabolism.

Effects of ROS on cell metabolism have been well documented in a varietyof species. These include not only a role in apoptosis, but also in theinduction of host defense genes and mobilization of ion transportsystems. ROS are implicated in redox signaling, also known as oxidativesignaling. In addition, ROS are implicated in cellular activity to avariety of inflammatory responses including cardiovascular disease. Ingeneral, harmful effects of ROS on a cell include: (a) damage of DNA;(b) oxidation of polyunsaturated fatty acids in lipids (lipidperoxidation); (c) oxidation of amino acids in proteins; and (d)inactivation of certain enzymes by oxidation of co-factors.

Hydrogen peroxide is generated in a variety of ways within the cell.Enzymes such as the monoamine oxidases produce hydrogen peroxide as aproduct of their enzymatic activity. Hydrogen peroxide can also beformed by interconversion of other reactive oxygen species such as thatproduced by superoxide dismutase when reacting with superoxide.

Current hydrogen peroxide detection techniques, such as AmplexRed, haveseveral limitations including: requiring horseradish peroxidase,requiring an enzyme coupled assay, instability in the presence of thiolssuch as DTT, 2-mercaptoethanol, etc., and instablity at pH (>8.5).

SUMMARY

In one embodiment, the invention provides a compound according toFormula (I)

wherein

R₁ is boronic acid or a borate ester;

each R₄, R₅ and R₇ is independently selected from H, halo, methyl, andtrifluoromethyl; and

L₁ is a linker.

The present invention also provides a compound according to Formula(II):

wherein

R₁₁ is boronic acid or a borate ester;

R² is —(CH₂)_(n)-T or C₁₋₅ alkyl;

R⁶ is selected from the group consisting of —H, —OH, —NH₂—OC(O)R or—OCH₂OC(O)R;

R⁸ is selected from the group consisting of

H or lower cycloalkyl;

wherein R³ and R⁴ are both H or both C₁₋₂ alkyl;

n is 0 to 3;

each R is independently a C₁₋₇ alkyl;

T is aryl, heteroaryl, substituted aryl, substituted heteroaryl orcycloalkyl;

L₂ is a linker;

and the dashed bonds indicate the presence of an optional ring which maybe saturated or unsaturated.

In addition, the present invention provides a compound according toFormula (III):

wherein

R₁₁ is boronic acid or a borate ester;

R² is —(CH₂)_(n)-T or C₁₋₅ alkyl;

R⁶ is selected from the group consisting of —H, —OH, —NH₂—OC(O)R or—OCH₂OC(O)R;

R⁸ is selected from the group consisting of

H or lower cycloalkyl;

wherein R³ and R⁴ are both H or both C₁₋₂ alkyl;

n is 0 to 3;

each R is independently a C₁₋₂ alkyl;

T is aryl, heteroaryl, substituted aryl, substituted heteroaryl orcycloalkyl;

L₃ is a linker;

and the dashed bonds indicate the presence of an optional ring which maybe saturated or unsaturated.

In another embodiment, the invention provides a method of detectinghydrogen peroxide in a cell, wherein the cell is contacted with acompound according to Formula (I), (II) or (III) and a luciferasereaction mixture, and bioluminescence is measured thereby detecting thepresence of hydrogen peroxide in the cell.

In yet another embodiment, the invention provides a method of detectinghydrogen peroxide in a cell, wherein the cell is contacted with acompound according to Formula (I), (II) or (III) to form an incubationmixture. At least a portion of the incubation mixture is transferred toa second reaction vessel, and a luciferase reaction mixture is added tothe second reaction vessel. Bioluminescence is then measured therebydetecting the presence of hydrogen peroxide in the cell.

In a further embodiment, the invention provides a method of detectinghydrogen peroxide in a sample wherein a sample is contacted with acompound according to Formula (I), (II), or (III) and a luciferasereaction mixture and D-cysteine. Bioluminescence is then measuredthereby detecting the presence of hydrogen peroxide in the sample.

In yet another embodiment, the invention provides a method ofdetermining the effect of a test compound on the presence or amount ofhydrogen peroxide in a sample wherein the sample is contacted with atest compound. A compound according to Formula (I), (II) or (III) and aluciferase reaction mixture is added, and bioluminescence is measured.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1, illustrates various luciferin borates.

FIG. 2, illustrates various benzothiazoles.

FIG. 3 shows sensitive detection of hydrogen peroxide using PBI 4759.

FIG. 4 shows addition of a reduction reagent can reduce background thatmay be seen with the luciferin borates such as PBI 4472.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compounds useful for detection ofhydrogen peroxide and methods of using same.

DEFINITIONS

As used herein, the following terms and expressions have the indicatedmeanings. It will be appreciated that the compounds of the presentinvention contain asymmetrically substituted carbon atoms and may beisolated in optically active or racemic forms. It is well known in theart how to prepare optically active forms such as by resolution ofracemic forms or by synthesis from optically active starting materials.All chiral, diastereomeric, racemic forms and all geometric isomericforms of a structure are part of this invention.

Specific values listed below for radicals, substituents, and ranges, arefor illustration only; they do not exclude other defined values or othervalues within defined ranges for the radicals and substituents.

As used herein, the term “substituted” is intended to indicate that oneor more (e.g., 1, 2, 3, 4, or 5; in some embodiments 1, 2, or 3; and inother embodiments 1 or 2) hydrogens on the group indicated in theexpression using “substituted” is replaced with a selection from theindicated group(s), or with a suitable group known to those of skill inthe art, provided that the indicated atom's normal valency is notexceeded, and that the substitution results in a stable compound.Suitable indicated groups include, e.g., alkyl, alkenyl, alkynyl,alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl,heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, alkylamino,dialkylamino, trifluoromethylthio, difluoromethyl, acylamino, nitro,trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo,alkylthio, alkylsulfinyl, alkylsulfonyl, arylsulfinyl, arylsulfonyl,heteroarylsulfinyl, heteroarylsulfonyl, heterocyclesulfinyl,heterocyclesulfonyl, phosphate, sulfate, hydroxylamine, hydroxyl(alkyl)amine, and cyano. Additionally, the suitable indicated groups caninclude, e.g., —X, —R, —O⁻, —OR, —SR, —S⁻, —NR₂, —NR₃, ═NR, —CX₃, —CN,—OCN, —SCN, —N═C═O, —NCS, —NO, —NO₂, ═N₂, —N₃, NC(═O)R, —C(═O)R,—C(═O)NRR, —S(═O)₂O⁻, —S(═O)₂OH, —S(═O)₂R, —OS(═O)₂OR, —S(═O)₂NR,—S(═O)R, —OP(═O)O₂RR, —P(═O)O₂RR, —P(═O)(O)₂, —P(═O)(OH)₂, —C(═O)R,—C(═O)X, —C(S)R, —C(O)OR, —C(O)O, —C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR,—C(S)NRR, —C(NR)NRR, where each X is independently a halogen (“halo”):F, Cl, Br, or I; and each R is independently H, alkyl, aryl, heteroaryl,heterocycle, a protecting group or prodrug moiety. As would be readilyunderstood by one skilled in the art, when a substituent is oxo (═O) orthioxo (═S), or the like, then two hydrogen atoms on the substitutedatom are replaced.

As used herein, the term “alkyl” refers to a branched, unbranched, orcyclic hydrocarbon having, for example, from 1 to 30 carbon atoms, andoften 1 to 12, or 1 to about 6 carbon atoms. Examples include, but arenot limited to, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl,2-methyl-1-propyl, 2-butyl, 2-methyl-2-propyl (t-butyl), 1-pentyl,2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl,3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl,2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl,3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl,3,3-dimethyl-2-butyl, hexyl, octyl, decyl, dodecyl, and the like. Thealkyl can be unsubstituted or substituted. The alkyl can also beoptionally partially or fully unsaturated. As such, the recitation of analkyl group includes both alkenyl and alkynyl groups. The alkyl can be amonovalent hydrocarbon radical, as described and exemplified above, orit can be a divalent hydrocarbon radical (i.e., alkylene).

The term “alkenyl” refers to a monoradical branched or unbranchedpartially unsaturated hydrocarbon chain (i.e. a carbon-carbon, sp²double bond). In one embodiment, an alkenyl group can have from 2 to 10carbon atoms, or 2 to 6 carbon atoms. In another embodiment, the alkenylgroup has from 2 to 4 carbon atoms. Examples include, but are notlimited to, ethylene or vinyl, allyl, cyclopentenyl, 5-hexenyl, and thelike. The alkenyl can be unsubstituted or substituted.

The term “alkynyl” refers to a monoradical branched or unbranchedhydrocarbon chain, having a point of complete unsaturation (i.e. acarbon-carbon, sp triple bond). In one embodiment, the alkynyl group canhave from 2 to 10 carbon atoms, or 2 to 6 carbon atoms. In anotherembodiment, the alkynyl group can have from 2 to 4 carbon atoms. Thisterm is exemplified by groups such as ethynyl, 1-propynyl, 2-propynyl,1-butynyl, 2-butynyl, 3-butynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl,1-octynyl, and the like. The alkynyl can be unsubstituted orsubstituted.

The term “cycloalkyl” refers to cyclic alkyl groups of from 3 to 10carbon atoms having a single cyclic ring or multiple condensed rings.Such cycloalkyl groups include, by way of example, single ringstructures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, andthe like, or multiple ring structures such as adamantanyl, and the like.The cycloalkyl can be unsubstituted or substituted. The cycloalkyl groupcan be monovalent or divalent, and can be optionally substituted asdescribed above for alkyl groups. The cycloalkyl group can optionallyinclude one or more cites of unsaturation, for example, the cycloalkylgroup can include one or more carbon-carbon double bonds, such as, forexample, cyclohexene, 1,3-cyclohexadiene, 1,4-cyclohexadiene, and thelike.

The term “alkoxy” refers to the group alkyl-O—, where alkyl is asdefined herein. In one embodiment, alkoxy groups include, e.g., methoxy,ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy,n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like. The alkoxy can beunsubstituted or substituted.

As used herein, “aryl” or “Ar” refers to an aromatic hydrocarbon groupderived from the removal of one hydrogen atom from a single carbon atomof a parent aromatic ring system. The radical can be at a saturated orunsaturated carbon atom of the parent ring system. The aryl group canhave from 6 to 30 carbon atoms. The aryl group can have a single ring(e.g., phenyl) or multiple condensed (fused) rings, wherein at least onering is aromatic (e.g., naphthyl, dihydrophenanthrenyl, fluorenyl, oranthryl). Typical aryl groups include, but are not limited to, radicalsderived from benzene, naphthalene, anthracene, biphenyl, and the like.The aryl can be unsubstituted or optionally substituted, as describedabove for alkyl groups.

The term “halo” refers to fluoro, chloro, bromo, and iodo. Similarly,the term “halogen” refers to fluorine, chlorine, bromine, and iodine.

The term “haloalkyl” refers to alkyl as defined herein substituted by 1or more halo groups as defined herein, which may be the same ordifferent. In one embodiment, the haloalkyl can be substituted with 1,2, 3, 4, or 5 halo groups. In another embodiment, the haloalkyl can bysubstituted with 1, 2, or 3 halo groups. The term haloalkyl also includeperfluoro-alkyl groups. Representative haloalkyl groups include, by wayof example, trifluoromethyl, 3-fluorododecyl, 12,12,12-trifluorododecyl,2-bromooctyl, 3-bromo-6-chloroheptyl, 1H,1H-perfluorooctyl, and thelike. The haloalkyl can be optionally substituted as described above foralkyl groups.

The term “heteroaryl” is defined herein as a monocyclic, bicyclic, ortricyclic ring system containing one, two, or three aromatic rings andcontaining at least one nitrogen, oxygen, or sulfur atom in an aromaticring, and that can be unsubstituted or substituted, for example, withone or more, and in particular one to three, substituents, as describedabove in the definition of “substituted”. Typical heteroaryl groupscontain 2-20 carbon atoms in addition to the one or more heteroatoms.Examples of heteroaryl groups include, but are not limited to,2H-pyrrolyl, 3H-indolyl, 4H-quinolizinyl, acridinyl, benzo[b]thienyl,benzothiazolyl, 13-carbolinyl, carbazolyl, chromenyl, cinnolinyl,dibenzo[b,d]furanyl, furazanyl, furyl, imidazolyl, imidizolyl,indazolyl, indolisinyl, indolyl, isobenzofuranyl, isoindolyl,isoquinolyl, isothiazolyl, isoxazolyl, naphthyridinyl, oxazolyl,perimidinyl, phenanthridinyl, phenanthrolinyl, phenarsazinyl,phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl,pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl,pyridyl, pyrimidinyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolyl,quinoxalinyl, thiadiazolyl, thianthrenyl, thiazolyl, thienyl, triazolyl,tetrazolyl, and xanthenyl. In one embodiment the term “heteroaryl”denotes a monocyclic aromatic ring containing five or six ring atomscontaining carbon and 1, 2, 3, or 4 heteroatoms independently selectedfrom non-peroxide oxygen, sulfur, and N(Z) wherein Z is absent or is H,O, alkyl, aryl, or (C₁-C₆)alkylaryl. In another embodiment heteroaryldenotes an ortho-fused bicyclic heterocycle of about eight to ten ringatoms derived therefrom, particularly a benz-derivative or one derivedby fusing a propylene, trimethylene, or tetramethylene diradicalthereto.

The term “heterocycle” refers to a saturated or partially unsaturatedring system, containing at least one heteroatom selected from the groupoxygen, nitrogen, and sulfur, and optionally substituted with one ormore groups as defined herein under the term “substituted”. Aheterocycle can be a monocyclic, bicyclic, or tricyclic group containingone or more heteroatoms. A heterocycle group also can contain an oxogroup (═O) or a thioxo (═S) group attached to the ring. Non-limitingexamples of heterocycle groups include 1,3-dihydrobenzofuran,1,3-dioxolane, 1,4-dioxane, 1,4-dithiane, 2H-pyran, 2-pyrazoline,4H-pyran, chromanyl, imidazolidinyl, imidazolinyl, indolinyl,isochromanyl, isoindolinyl, morpholine, piperazinyl, piperidine,piperidyl, pyrazolidine, pyrazolidinyl, pyrazolinyl, pyrrolidine,pyrroline, quinuclidine, and thiomorpholine.

The term “heterocycle” can include, by way of example and notlimitation, a monoradical of the heterocycles described in Paquette, LeoA.; Principles of Modern Heterocyclic Chemistry (W. A. Benjamin, NewYork, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; The Chemistryof Heterocyclic Compounds, A Series of Monographs” (John Wiley & Sons,New York, 1950 to present), in particular Volumes 13, 14, 16, 19, and28; and J. Am. Chem. Soc. 1960, 82, 5566. In one embodiment,“heterocycle” includes a “carbocycle” as defined herein, wherein one ormore (e.g. 1, 2, 3, or 4) carbon atoms have been replaced with aheteroatom (e.g. O, N, or S).

Examples of heterocycles, by way of example and not limitation, include,dihydroypyridyl, tetrahydropyridyl (piperidyl), thiazolyl,tetrahydrothiophenyl, sulfur oxidized tetrahydrothiophenyl, pyrimidinyl,furanyl, thienyl, pyrrolyl, pyrazolyl, piperidinyl, 4-piperidonyl,pyrrolidinyl, 2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl,tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl,octahydroisoquinolinyl, azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl,2H,6H-1,5,2-dithiazinyl, thienyl, thianthrenyl, pyranyl,isobenzofuranyl, chromenyl, xanthenyl, phenoxathinyl, 2H-pyrrolyl,isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl,isoindolyl, 3H-indolyl, 1H-indazoly, purinyl, 4H-quinolizinyl,phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl,pteridinyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl,pyrimidinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, furazanyl,phenoxazinyl, isochromanyl, chromanyl, imidazolidinyl, imidazolinyl,pyrazolidinyl, pyrazolinyl, piperazinyl, indolinyl, isoindolinyl,quinuclidinyl, morpholinyl, oxazolidinyl, benzotriazolyl,benzisoxazolyl, oxindolyl, benzoxazolinyl, isatinoyl, andbis-tetrahydrofuranyl.

By way of example and not limitation, carbon bonded heterocycles arebonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5, or6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position 2,3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan,tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole,position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4,or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of anaziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6,7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of anisoquinoline. Carbon bonded heterocycles include 2-pyridyl, 3-pyridyl,4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl,5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl,5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl,6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, and the like.

By way of example and not limitation, nitrogen bonded heterocycles canbe bonded at position 1 of an aziridine, azetidine, pyrrole,pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine,2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline,3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole,position 2 of a isoindole, or isoindoline, position 4 of a morpholine,and position 9 of a carbazole, or β-carboline. In one embodiment,nitrogen bonded heterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl,1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.

The term “carbocycle” refers to a saturated, unsaturated or aromaticring having 3 to 8 carbon atoms as a monocycle, 7 to 12 carbon atoms asa bicycle, and up to about 30 carbon atoms as a polycycle. Monocycliccarbocycles typically have 3 to 6 ring atoms, still more typically 5 or6 ring atoms. Bicyclic carbocycles have 7 to 12 ring atoms, e.g.,arranged as a bicyclo [4,5], [5,5], [5,6] or [6,6] system, or 9 or 10ring atoms arranged as a bicyclo[5,6] or [6,6] system. Examples ofcarbocycles include cyclopropyl, cyclobutyl, cyclopentyl,1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, phenyl, spiryland naphthyl. The carbocycle can be optionally substituted as describedabove for alkyl groups.

The term “alkanoyl” or “alkylcarbonyl” refers to —C(═O)R, wherein R isan alkyl group as previously defined.

The term “acyloxy” or “alkylcarboxy” refers to —O—C(═O)R, wherein R isan alkyl group as previously defined. Examples of acyloxy groupsinclude, but are not limited to, acetoxy, propanoyloxy, butanoyloxy, andpentanoyloxy. Any alkyl group as defined above can be used to form anacyloxy group.

The term “alkoxycarbonyl” refers to —C(═O)OR (or “COOR”), wherein R isan alkyl group as previously defined.

The term “amino” refers to —NH₂. The amino group can be optionallysubstituted as defined herein for the term “substituted”.

The term “alkylamino” refers to —NR₂, wherein at least one R is alkyland the second R is alkyl or hydrogen.

The term “acylamino” refers to N(R)C(═O)R, wherein each R isindependently hydrogen, alkyl, or aryl.

The term “hydroxyalkyl” refers to an alkyl group substituted by —OH.

The term “alkylcarboxylic acid” refers to an alkyl group substituted by—COOH.

The term “interrupted” indicates that another group is inserted betweentwo adjacent carbon atoms (and the hydrogen atoms to which they areattached (e.g., methyl (CH₃), methylene (CH₂) or methine (CH))) of aparticular carbon chain being referred to in the expression using theterm “interrupted, provided that each of the indicated atoms' normalvalency is not exceeded, and that the interruption results in a stablecompound. Suitable groups that can interrupt a carbon chain include,e.g., with one or more non-peroxide oxy (—O—), thio (—S—), imino(—N(H)—), methylene dioxy (—OCH₂O—), carbonyl (—C(═O)—), carboxy(—C(═O)O—), carbonyldioxy (—OC(═O)O—), carboxylato (—OC(═O)—), imine(C≡NH), sulfinyl (SO) and sulfonyl (SO₂). Alkyl groups can beinterrupted by one ore more (e.g., 1, 2, 3, 4, 5, or about 6) of theaforementioned suitable groups. The site of interruption can also bebetween a carbon atom of an alkyl group and a carbon atom to which thealkyl group is attached.

The term “linker” refers to a (C₁-C₁₂)alkyl diradical that is optionallyinterrupted by one to four O atoms, N atoms, S atoms, or an optionallysubstituted aryl, heteroaryl, or heterocycle group.

The term “luciferase,” unless specified otherwise, refers to a naturallyoccurring, recombinant or mutant luciferase. The luciferase, ifnaturally occurring, may be obtained easily by the skilled person froman organism. If the luciferase is one that occurs naturally or is arecombinant or mutant luciferase, i.e. one which retains activity in aluciferase-luciferin reaction of a naturally occurring luciferase, itcan be obtained readily from a culture of bacteria, yeast, mammaliancells, insect cells, plant cells, or the like, transformed to express anucleic acid encoding the luciferase. Further, the recombinant or mutantluciferase can be derived from an in vitro cell-free system using anucleic acid encoding the luciferase. Luciferases are available fromPromega Corporation, Madison, Wis.

As used herein, “bioluminescence” or “luminescence” is light produced asa result of a reaction between an enzyme and a substrate that generateslight. Examples of such enzymes (bioluminescent enzymes) include fireflyluciferase, e.g. Photinus pyralis or Photinus pennslyvanica, clickbeetle luciferase, cypridina luciferase, and the like.

A “luciferase reaction mixture” contains a luciferase enzyme andmaterials that will allow the luciferase enzyme to generate a lightsignal. The materials needed, and the particular concentrations and/oramounts, of the materials needed to generate a luminescent signal willvary depending on the luciferase enzyme used as well as the type ofluciferase-based assay being performed. In general, for fireflyluciferase, these materials can include: ATP, a magnesium (Mg²⁺) salt,such as magnesium sulfate, a firefly luciferase enzyme, e.g, athermostable firefly luciferase, and a luciferin capable of generatinglight when the luciferin is used as a substrate for the fireflyluciferase. Often other materials will be added to the solutionincluding: a buffer to maintain the reaction at the proper pH, anadditive such as PRIONEX or Bovine serum albumin (BSA) to help maintainluciferase activity, reducing agents, detergents, esterases, salts,amino acids, e.g. D-cysteine, etc. An example luciferase reactionmixture would contain a thermostable firefly luciferase, MgSO₄, ATP,Tergitol NP-9, and Tricine. An alternative example luciferase reactionmixture would include Oplophorus luciferase, e.g., NanoLuc luciferase,buffer, e.g., Tris—Cl or Tris base, and optionally a backgroundreduction agent, e.g., TCEP.

Compounds

In one aspect, the invention provides compounds according to Formula(I):

wherein

R₁ is boronic acid or a borate ester;

each R₄, R₅ and R₇ is independently selected from H, halo, methyl, andtrifluoromethyl; and

L₁ is a linker.

In certain embodiments, R₁ is a borate ester. For example, R₁ can be—B(OR₆)₂; wherein each R₆ is independently selected from H and C₁₋₄alkyl.

R₁ may also be

wherein each R₁₂ and R₁₃ is independently selected from H, C₁₋₄ alkyl,CF₃, phenyl or substituted phenyl. Alternatively, R₁₂ and R₁₃ togethercan be an alkyl ring having from 3-7 carbons or can be replaced by afused 6-membered aromatic ring.

In addition, R₁ may be

wherein each R₁₄, R₁₅ and R₁₆ is independently selected from H, C₁₋₄alkyl, CF₃, phenyl and substituted phenyl. Alternatively, both R₁₅together can form an alkyl ring having from 3-7 carbons; R₁₄ and R₁₅together or R₁₅ and R₁₆ together can be an alkyl ring having from 3-7carbon atoms or can be replaced by a 6-membered aromatic ring.

In certain embodiments, L₁ is

A is —C₆(R₁₀)₄- or —(CR₁₁═CR₁₁)_(n)— or a direct bond or —O—(C₆(R₁₀)₄-or —S—C₆(R₁₀)₄- or —NR′ —C₆(R₁₀)₄; R′ is H or C₁₋₄ alkyl; each R₃ isindependently halo, H, C₁₋₄ alkyl, C₁₋₄ hydroxyalkyl, or C₁₋₄alkylcarboxylic acid; each R₁₀ is independently H, halo, CH₃, OCH₃, orNO₂; each R₁₁ is independently H or CH₃; n is 1 or 2; and X is selectedfrom —O—,

In some embodiments, -L₁-R₁ is

In additional embodiments, -L₁-R₁ is

where A is —O—(C₆H₄)— and X is —O—. In other embodiments, -L₁-R₁ is

where A is a direct bond and X is —O—.

In certain embodiments, -L₁-R₁ is

where A is —O—(C₆H₄)— and X is —NHCO₂—. In certain embodiments, -L₁-R₁is

where A is —O—(C₆H₄)— and X is —NHC(O)CH₂—.

Suitable compounds according to Formula (I) include those shown in FIG.2 and below:

In another embodiment, the present invention provides a compound ofFormula (II):

or Formula (III):

wherein

R₁₁ is boronic acid or a borate ester;

R² is —(CH₂)_(n)-T or C₁₋₅ alkyl;

R⁶ is selected from the group consisting of —H, —OH, —NH₂—OC(O)R or—OCH₂OC(O)R;

R⁸ is selected from the group consisting of

H or lower cycloalkyl;

wherein R³ and R⁴ are both H or both C₁₋₂ alkyl;

n is 0 to 3;

each R is independently a C₁₋₇ alkyl;

T is aryl, heteroaryl, substituted aryl, substituted heteroaryl orcycloalkyl;

L₂ or L₃ is a linker;

and the dashed bonds indicate the presence of an optional ring which maybe saturated or unsaturated.

In certain embodiments, R₁₁ is a borate ester. For example, R₁₁ can be—B(OR₇)₂; wherein each R₇ is independently selected from H and C₁₋₄alkyl. R₁₁ may also be

wherein each R₁₂ and R₁₃ is independently selected from H, C₁₋₄ alkyl,CF₃, phenyl or substituted phenyl. Alternatively, R₁₂ and R₁₃ togethercan be an alkyl ring having from 3-7 carbons or can be replaced by afused 6-membered aromatic ring.

In addition, R₁₁ may be

wherein each R₁₄, R₁₅ and R₁₆ is independently selected from H, C₁₋₄alkyl, CF₃, phenyl and substituted phenyl. Alternatively, both R₁₅together can form an alkyl ring having from 3-7 carbons; R₁₄ and R₁₅together or R₁₅ and R₁₆ together can be an alkyl ring having from 3-7carbon atoms or can be replaced by a 6-membered aromatic ring.

In certain embodiments, L₂ is

A is —C₆(R₁₀)₄- or —(CR₂₁═CR₂₁)_(n)— or —O—C₆(R₁₀)₄- or —S—C₆(R₁₀)₄- or—NR′ —C₆(R₁₀)₄- or a direct bond; R′ is H or C₁₋₄ alkyl; each R₃ isindependently halo, H, C₁₋₄ alkyl, C₁₋₄ hydroxyalkyl, or C₁₋₄alkylcarboxylic acid; each R₁₀, is independently H, halo, CH₃, OCH₃, orNO₂; each R₂₁ is independently H or CH₃; n is 1 or 2; and X is selectedfrom a direct bond, —C(O)—, and —C(O)NR₂₂, where R₂₂ is H or C₁₋₄ alkyl.

In some embodiments, -L₂-R₁₁ is

In certain embodiments, L₃ is

wherein A is —C₆(R₁₀)₄-, or —(CR₂₁═CR₂₁)_(n)—; each R₃ is independentlyhalo, H, C₁₋₄ alkyl, C₁₋₄ hydroxyalkyl, or C₁₋₄ alkylcarboxylic acid;each R₁₀, is independently H, halo, CH₃, OCH₃, or NO₂; each R₂₁ isindependently H or CH₃; and n is 1 or 2.

In certain embodiments, -L₃-R₁₁ is

In some embodiments, R² is

or C₂₋₅ alkyl; each X is independently —S—, —O— or —NH—; Z is —CH— or—N—; Y is —H or —OH; W is —NH₂, halo, —OH, —NHC(O)R, —CO₂R; and R isC₁₋₇ alkyl.

In some embodiments, R² is

and X is O or S. In other embodiments, R² is C₂₋₅ straight-chain alkyl.In certain embodiments, R⁸ is

lower cycloalkyl or H, wherein R³ and R⁴ are both H or C₁₋₂ alkyl. Inother embodiments, R⁸ is benzyl.

Suitable compounds according to Formula (II) include:

Synthesis of Compounds

Compounds described herein may be synthesized using a variety ofmethods. Exemplary syntheses are generalized in Schemes 1 and 2 below.

The synthesis of a pinacol borate of aromaticbenzyl-6-O-2-cyanobenzothiazole can be accomplished by two steps.Typically, borate pinacol ester 4-aromatic methyl alcohol is convertedinto pinacol borate 4-aromatic methyl bromide with Ph₃P and CBr₄, andthen reacted with 2-cyano-6-hydroxybenzothiazole in the presence ofK₂CO₃ to obtain the desired compound. The synthesis of pinacol borate ofally-6-O-2-cyanobenzothiazole can be completed by direct alkylation of6-hydroxy-2-cyanobenzothiazole with pinacol borate ally iodide. (Scheme1)

Typically, pinacol borate aromatic methyl alcohol is converted to itscarbonochloridate with triphosgen in the presence of pyridine and thenreacted with 6-amino-2-cyanobenzothiazole to obtain the targetmolecules.

Borate coelenterazine derivatives generally may be synthesized byalkylating coelenterazine with pinacol borate 4-aromatic methyl bromideto give O-alkylated coelenterazine borate and C-alkylated coelenterazineborate. (Scheme 3)

As can be appreciated by the skilled artisan, alternative methods ofsynthesizing the compounds of the formulae herein will be evident tothose of ordinary skill in the art. Additionally, the various syntheticsteps may be performed in an alternate sequence or order to give thedesired compounds. Synthetic chemistry transformations and protectinggroup methodologies (protection and deprotection) useful in synthesizingthe compounds described herein are known in the art and include, forexample, those such as described in R. Larock, Comprehensive OrganicTransformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons(1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995), and subsequent editions thereof.

Methods of Use

In an aspect, the invention provides a method of detecting hydrogenperoxide. In one embodiment, a sample is contacted with a compoundaccording to Formula (I), (II) or (III) described herein to form a firstmixture. At least a portion of the first mixture is contacted with aluciferase reaction mixture and D-cysteine. Bioluminescence is detectedthereby detecting the presence of hydrogen peroxide.

In another embodiment of the present invention, cells are contacted witha compound according to Formula (I), (II) or (III) described herein, aluciferase reaction mixture is added to the contacted cells, andbioluminescence is detected. In certain embodiments, D-luciferin is alsoadded to the contacted cells.

In another aspect, the invention provides a method for determining theeffect of a test compound or test condition on the presence or amount ofhydrogen peroxide in a sample, cell or animal. In one embodiment, thesample, cells or animal are contacted with a test compound or testcondition prior to being contacted with a compound according to Formula(I), (II) or (III) described herein and a luciferase reaction mixture.The effect of the test compound or test condition on the presence oramount of hydrogen peroxide in the sample or cells is determined bydetecting bioluminescence. Suitably, the test condition may be a changein temperature, oxygen tension, or ionic strength or an osmotic change.

The reagents may be added sequentially or simultaneously. If thereagents are added simultaneously, they may be in a single solution ormultiple solutions.

The signal may be quantified if desired. The signal may be compared to astandard curve. The intensity of the signal is a function of thepresence of amount of hydrogen peroxide in the sample. The signal mayalso be compared to a control.

The present invention may be used to determine the presence or amount ofhydrogen peroxide in cells grown in culture medium or in cells withinanimals, e.g., living animals. For research purposes, for measurementsin cells in animals, a compound according to Formula (I), (II) or (III)described herein is administered, e.g., injected into the animal oradded to an aqueous solution, e.g., water, or food consumed by theanimal, to the animal. Conversion of the compound to a product that is aluciferase substrate may be detected by bioluminescence mediated byluciferase expressed in cells in the animal, e.g., whole animal imagingof a transgenic animal (e.g., mice, rats, and marmoset monkeys) byluciferase administered to the animal, e.g., injected into the animal,or by collecting physiological fluids, e.g., blood, plasma, urine, andthe like, or tissue samples, and combining those with a luciferasereaction mixture.

Cells may be eukaryotic cells, e.g., yeast, avian, plant, insect ormammalian cells, including but not limited to human, simian, murine,canine, bovine, equine, feline, ovine, caprine or swine cells, orprokaryotic cells, or cells from two or more different organisms, orcell lysates or supernatants thereof. The cells may have beengenetically modified via recombinant techniques. In certain aspects, thecell may be in an animal, e.g., transgenic animals, or physiologicalfluid, e.g., blood, plasma, urine, mucous secretions or the like.Destruction of the cells is not required as the media can be sampled.

In addition, for any of the bioluminogenic assays described herein,other reagents may be added to reaction mixtures, including but notlimited to those that inhibit or prevent inactivation of luciferase, orotherwise extend or enhance the bioluminescent signal.

The bioluminescence generated may be compared to a control. Suitablecontrols lack one or more of the necessary components or conditions foreither the reaction between the compound and hydrogen peroxide or theluciferase reaction. Such components or conditions include, but are notlimited to, co-factors, enzyme, temperature, and inhibitors.

Suitable substrates include, but are not limited to, compounds ofFormulas (I) or (II) described herein.

Certain substrates may be particularly advantageous for use in variousembodiments of this invention. For example, certain substrates may bebetter suited to use in vitro and others may be better suited to use invivo. As would be recognized by one of ordinary skill in the art, notall borates would be suitable for use in the methods of the presentinvention. For example, small changes in the compounds can affect thereactivity toward hydrogen peroxide or the ability to work in certainassay conditions.

The invention is further described by the following non-limitingexamples.

EXAMPLES Example 16-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)benzo-[d]thiazole-2-carbonitrile(PBI 4472)

Synthesis of2-(4-(bromomethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(1203-32)

4-hydroxymethylphenylboronic acid, pinacol ester (1.08 g, 4.61 mmol) wasdissolved in THF (20 ml) together with triphenylphosphine (2.42 g, 9.23mmol). The reaction mixture was cooled in an ice-water bath, and carbontetrabromide (3.06 g, 9.23 mmol) was added portion wise. After stirringfor 4 hours at room temperature, the reaction mixture was poured intowater and extracted with ethyl acetate. The organic layer was combinedand dried by sodium sulfate. After filtration, the solvent wasevaporated, and the residue was purified by flash chromatography to givethe product as a white solid (1.72 g, 92%). ¹H NMR (300 MHz, CD₂Cl₂, δ):7.62 (d, J=6.0 Hz, 2H), 7.32 (d, J=6.0 Hz, 2H), 4.58 (d, 2H), 1.34 (s,9H); MS (ESI) m/z 297.0.

Synthesis of6-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)benzo-[d]thiazole-2-carbonitrile(PBI 4472)

A mixture of 6-hydroxybenzothiazole (0.5 g, 2.84 mmol), potassiumcarbonate (0.78 g, 5.68 mmol) and potassium iodide (0.94 g, 5.68 mmol)in acetonitrile was heated to reflux for 1 hour.2-(4-(bromomethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (0.93g, 3.12 mmol) was added, and the reaction mixture was refluxedovernight. After cooling down, the suspension was extracted with ethylacetate/water and the residue was purified with flash chromatography togive the product as a white solid (0.8 g, 72%). ¹H NMR (300 MHz, CD₂Cl₂,δ): 8.11 (d, J=9.0 Hz, 1H), 7.81 (d, J=9.0 Hz, 2H), 7.47 (m, 3H), 7.34(dd, J=9.0 Hz, 1H), 5.22 (s, 2H), 1.35 (s, 9H); MS (ESI) m/z 393.2.

Example 2 (4-(4-(((2-cyanobenzo[d]thiazol-6-yl)oxy)methyl)phenyl)boronicacid (PBI 4452)

To a solution of6-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)benzo[d]thiazole-2-carbonitrile(PBI 4472) (0.26 g, 0.66 mmol) in acetone (40 ml), a suspension ofsodium periodate (0.43 g, 1.99 mmol), ammonium acetate (0.11 g, 1.33mmol) in water (40 ml) was added at room temperature. The thicksuspension was allowed to stir at room temperature for 18 hours. Allsolvent was evaporated, and the residue was dissolved in DMF. Thesuspension was centrifuged, and the clean solution was purified byprep-HPLC with acetonitrile/10% ammonium acetate to give the product asa white crystal (0.15 g, 72%). ¹H NMR (300 MHz, DMSO, δ): 8.15 (d, J=9.0Hz, 1H), 8.04 (s, 2H), 7.97 (d, J=3.0 Hz, 1H), 7.80 (d, J=9.0 Hz, 2H),7.41 (m, 3H), 5.23 (s, 2H); MS (ESI) m/z 311.1.

Example 36-((3-chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)-benzo[d]thiazole-2-carbonitrile(PBI 4595)

This compound was made following the same procedures as PBI 4472(Example 1).

Example 4(2-chloro-4-(((2-cyanobenzo[d]thiazol-6-yl)oxy)methyl)phenyl)boronicacid (PBI 4470)

This compound was made following the same procedure as PBI 4452 (Example2). ¹H NMR (300 MHz, DMF, 6): 8.43 (s, 2H), 8.22 (d, J=9.0 Hz, 1H), 8.07(m, 1H), 7.56 (m, 2H), 7.47 (m, 2H), 5.33 (s, 2H); MS (ESI) m/z 345.1.

Example 56-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)naphthalen-1-yl)methoxy)benzo[d]thiazole-2-carbonitrile(PBI 4480)

Synthesis of4-(4,4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-naphthoic acid(1203-33)

A mixture of 4-bromo-1-naphthoic acid (4.12 g, 16.43 mmol),bis(pinacolato)diborane (4.38 g, 17.25 mmol), cesium fluoride (7.49 g,49.28 mmol), triphenylphosphine (0.86 g, 3.29 mmol) in acetonitrile wasadded palladium acetate (0.37 g, 1.64 mmol). The reaction mixture wasrefluxed overnight. After cooling down, it was filtered through Celiteand extracted with ethyl acetate/water. The organic layer was collectedand dried over sodium sulfate. After filtration, solvent was removed andthe residue was purified by flash chromatography to give the product asa white solid (2.5 g, 51%). ¹H NMR (300 MHz, DMSO, δ): 8.76 (m, 1H),8.69 (m, 1H), 8.02 (m, 2H), 7.61 (m, 1H), 1.28 (s, 12H).

Synthesis of(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)naphthalen-1-yl)methanol(1203-44)

4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-naphthoic acid (2.5 g,8.39 mmol) was dissolved in anhydrous THF. The solution was cooled downin an ice-water bath. Borane solution (1.00M in THF, 25 ml, 25 mmol) wasadded dropwise. After addition, the reaction was stirred at roomtemperature for 4 hours. Methanol (20 ml) was added with ice-water batchto quench the reaction until no gas was produced. The solvent wasevaporated, and the residue was extracted with ethyl acetate/water togive the product as a white solid (1.79 g, 75%). ¹H NMR (300 MHz,CD₂Cl₂, δ): 8.80 (m, 1H), 8.10 (m, 1H), 8.04 (m, 1H), 7.57 (m, 3H), 5.18(s, 2H), 1.43 (s, 12H).

Synthesis of2-(4-(bromomethyl)naphthalen-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(1203-47)

This compound was made following the same procedure as 1203-32 (Example1). ¹H NMR (300 MHz, CD₂Cl₂, δ): 8.82 (m, 1H), 8.19 (m, 1H), 8.00 (m,1H), 7.60 (m, 3H), 5.02 (s, 2H), 1.43 (s, 12H).

Synthesis of64(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)naphthalen-1-yl)methoxy)benzo[d]thiazole-2-carbonitrile(PBI 4480)

This compound was made following the same procedure as PBI 4472 (Example1). ¹H NMR (300 MHz, CD₂Cl₂, δ): 8.85 (m, 1H), 8.11 (d, 1H), 8.06 (m,2H), 7.09 (m, 4H), 7.38 (m, 1H), 5.35 (s, 2H), 1.42 (s, 12H); MS (ESI)m/z 443.2.

Example 66-((3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)benzo[d]thiazole-2-carbonitrile (PBI 4481)

Synthesis of2-(4-(bromomethyl)-2-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(1203-40)

The compound was made following the same procedure as 1203-44 (Example5). ¹H NMR (300 MHz, CD₂Cl₂, δ): 7.70 (m, 1H), 7.15 (m, 1H), 7.06 (m,1H), 4.71 (s, 2H), 1.35 (s, 12H); MS (ESI) m/z 253.2.

Synthesis of2-(4-(bromomethyl)-2-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(1203-45)

The compound was made following the same procedure as 1203-32 (Example1). ¹H NMR (300 MHz, CD₂Cl₂, δ): 7.71 (m, 1H), 7.68 (m, 1H), 7.09 (m,1H), 4.49 (s, 2H), 1.35 (s, 12H); FNMR: 102.97; MS (ESI) m/z 315.1.

Synthesis of6-((3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)benzo[d]thiazole-2-carbonitrile(PBI 4481)

The compound was made following the same procedure as PBI 4472 (Example1). ¹H NMR (300 MHz, CD₂Cl₂, δ): 8.12 (d, J=9.0 Hz, 1H), 7.77 (m, 1H),7.45 (d, J=3.0 Hz, 1H), 7.35 (dd, J=3.0 Hz, 9.0 Hz), 7.26 (m, 1H), 7.17(m, 1H), 5.21 (s, 2H), 1.36 (s, 12H); MS (ESI) m/z 411.2.

Example 76-(1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)ethoxy)benzo[d]thiazole-2-carbonitrile(PBI 4513)

Synthesis of1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)ethanol(1203-59)

4-acetylphenyl boronic acid (3.0 g, 12.2 mmol) was dissolved inanhydrous ethanol (30 ml) and cooled in an ice/water bath. NaBH₄ (1.15g, 30.5 mmol) was added at once as a solid. After stirring overnight atroom temperature, the solution was cooled in an ice-water bath andtreated with 1N HCl (20 ml). The mixture was then extracted with ethylacetate, dried over sodium sulfate, filtered and concentrated undervacuum. The crude product obtained was purified with flashchromatography to give the product as a white solid (2.98 g, 99%). ¹HNMR (300 MHz, CD₂Cl₂, δ): 7.75 (m, 2H), 7.39 (m, 2H), 4.90 (q, J=6.0Hz), 1.47 (d, J=6.0 Hz), 1.32 (s, 12H).

Synthesis of2-(4-(1-bromoethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(1203-64)

The compound was made following the same procedure as 1203-32 (Example1). ¹H NMR (300 MHz, CD₂Cl₂, δ): 7.76 (m, 2H), 7.45 (m, 2H), 5.25 (q,J=6.0 Hz), 2.05 (d, J=6.0 Hz), 1.32 (s, 12H); MS (ESI) m/z 311.2.

Synthesis of6-(1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)ethoxy)benzo[d]thiazole-2-carbonitrile (PBI 4513)

The compound was made following the same procedure as PBI 4472 (Example1). ¹H NMR (300 MHz, CD₂Cl₂, δ): 8.03 (m, 1H), 7.76 (m, 2H), 7.44 (m,2H), 7.28 (m, 2H), 5.43 (q, J=6.0 Hz, 1H), 1.69 (d, J=6.0 Hz, 3H), 1.32(s, 12H); MS (ESI) m/z 407.2.

Example 86-((3-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)benzo[d]thiazole-2-carbonitrile (PBI 4512)

Synthesis of3-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid(1203-70)

The compound was made following the same procedure as 1203-33 (Example5). ¹H NMR (300 MHz, DMSO, δ): 7.74 (m, 1H), 7.66 (m, 1H), 7.57 (m, 1H),3.90 (s, 3H), 1.35 (s, 12H); MS (ESI) m/z 277.6.

Synthesis of(3-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methanol(1203-79)

The compound was made following the same procedure as 1203-44 (Example5) and used in the next step without purification.

Synthesis of2-(4-(bromomethyl)-2-methoxyphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(1203-82)

The compound was made following the same procedure as 1203-32(Example 1) and used in the next step without purification.

Synthesis of6-((3-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)benzo[d]thiazole-2-carbonitrile(PBI 4512)

The compound was made following the same procedure as 1203-36 (Example1). ¹H NMR (300 MHz, CD₂Cl₂, δ): 8.11 (d, J=9.0 Hz, 1H), 7.69 (d, J=9.0Hz, 1H), 7.47 (d, J=3.0 Hz, 1H), 7.35 (m, 1H), 7.04 (m, 1H), 6.98 (m,1H), 5.20 (s, 2H), 3.84 n(s, 3H), 1.34 (s, 12H).

Example 9 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl(2-cyanobenzo[d]thiazol-6-yl)carbamate (PBI 4579)

Triphosgen (0.26 g, 0.77 mmol) and 4-hydroxymethylphenylboronic acid,pinacol ester (0.5 g, 2.14 mmol) were dissolved in THF (20 ml) in anice-water bath. Pyridine (0.35 ml, 4.28 mmol) was added dropwise, andthe reaction mixture was stirred at 0° C. until thin layerchromatography showed the disappearance of starting material. Afterbring the temperature to room temperature, the reaction mixture wasextracted with dichloromethane/water. The organic layer was collectedand dried over sodium sulfate. After filtration, solvent was removed andthe residue was purified by flash chromatography to give the product asa white solid (0.1 g, 11%). ¹H NMR (300 MHz, CD₂Cl₂, δ): 8.43 (m, 1H),8.10 (m, 1H), 7.78 (m, 2H), 7.41 (m, 2H), 7.26 (m, 1H), 5.25 (s, 2H),1.32 (s, 12H); MS (ESI) m/z 436.1.

Example 106-((4-(5,5-dimethyl-1,3,2-dioxaborinan-2-yl)benzyl)oxy)benzo[d]thiazole-2-carbonitrile(PBI 4578)

A mixture of PBI 4452 (Example 2) (20 mg, 0.065 mmol) and neopentylglycol (34 mg, 0.32 mmol) in toluene (50 ml) was heated to reflux inDean-Stark apparatus. The reaction was cooled down after 16 hours.Toluene was evaporated, and the residue was purified by flashchromatography to give the product as a white solid (10 mg, 41%). ¹H NMR(300 MHz, CD₂Cl₂, δ): 8.11 (d, J=9.0 Hz, 1H), 7.82 (d, J=6.0 Hz, 2H),7.46 (m, 3H), 7.34 (dd, J=6.0 Hz, 3.0 Hz, 1H), 5.20 (s, 2H), 3.78 (s, s,4H), 1.03 (s, 6H).

Example 11(E)-6-((3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)allyl)oxy)-benzo[d]thiazole-2-carbonitrile(PBI 4458)

A reaction vial was charged with tetrabutylammonium2-cyanobenzo[d]thiazol-6-olate (209 mg, 1.19 mmol),(E)-2-(3-iodoprop-1-en-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(386 mg, 1.31 mmol) and 15 mL of dry dichloromethane. The vial wassealed, and the solution was heated in an oil bath at 80° C. overnight(˜16 hours). The crude reaction mixture was added to 1 gram of celite,and the solvent was evaporated under vacuum. The product was purified bysilica gel chromatography using an increasing gradient of ethyl acetatein dichloromethane as eluent. This gave 166 mg of(E)-6-((3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)allyl)oxy)benzo[d]thiazole-2-carbonitrileas a colorless oil that crystallized to a white solid upon standing atambient temperature.

Example 12 Synthesis of8-benzyl-2-(furan-2-ylmethyl)-6-phenyl-3-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)imidazo[1,2-a]pyrazine

Synthesis of8-benzyl-2-(furan-2-ylmethyl)-6-phenyl-3((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)imidazo[1,2-a]pyrazine(4759). To a solution of8-benzyl-2-(furan-2-ylmethyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one(50 mg, 0.13 mmol) was added2-(4-(bromomethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (47mg, 0.16 mmol), potassium carbonate (27 mg, 0.20 mmol) and potassiumiodide (33 mg, 0.20 mmol). The reaction mixture was stirred at roomtemperature for 1 h and heat to 40° C. until HPLC shows the completionof the reaction. After cooling down, the reaction mixture was extractedwith ethyl acetate/water. The organic layer was collected and dried overmagnesium sulfate. After evaporation, the residue was purified withflash chromatography to give the product as yellow solid (30 mg, 49%).¹H NMR (300 MHz, CD₂Cl₂, δ): 7.79-7.23 (m, 18H), 7.32 (d, J=6.0 Hz, 2H),5.11 (s, 2H), 4.53 (s, 2H), 4.15 (s, 2H), 1.53 (s, 12H); MS (ESI) m/z597.41.

Prophetic Example 13 Synthesis of8-benzyl-2-(furan-2-ylmethyl)-6-phenyl-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)imidazo[1,2-a]pyrazin-3(2H)-one

Synthesis of8-benzyl-2-(furan-2-ylmethyl)-6-phenyl-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)imidazo[1,2-a]pyrazin-3(2H)-one.To a solution of8-benzyl-2-(furan-2-ylmethyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one,2-(4-(bromomethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane,potassium carbonate and potassium iodide (33 mg, 0.20 mmol) is added.The reaction mixture is stirred at room temperature and is heated to 40°C. until HPLC shows the completion of the reaction.

Example 14 Generation of Bioluminescence Upon Treatment with Peroxide

In this example, luciferin borates were incubated with hydrogenperoxide, and luciferin generation was measured using a light reactioncatalyzed by luciferase. An increase in light output (bioluminescence)as a result of treatment of the luciferin borates with hydrogen peroxideindicates generation of luciferin as a result of the reaction withhydrogen peroxide.

1M phosphate solutions (either 6.8 g of KH₂PO₄ (Fisher) or 11.4 g ofK₂HPO₄ (Sigma) were dissolved in Nanopure water to 1M) were diluted 1:5with Nanopure water to produce 200 mM phosphate solutions. 200 mMphosphate buffers at pH 6.87, 7.41, 7.73, 8.04, and 9.18 were prepared.4 mg/ml solutions of PBI 3048, 4013, 4424 and 4425 (FIG. 1) in DMSO(Fluka 41641) were made from solid powder.

Into two 96-well white, solid bottom luminometer plates (Promega Z3291)(labeled “A” and “B”), 50 μl of 200 mM phosphate buffer pH 6.87 wasadded to wells A1-H2; 50 μl of 200 mM phosphate pH 7.41 was added towells A3-H4; 50 μl of 200 mM phosphate buffer pH 7.73 was added to wellsA5-H6; 50 μl of 200 mM phosphate buffer pH 8.04 was added to wellsA7-H8; and 50 μm of 200 mM phosphate buffer pH 9.18 was added to wellsA9-H10.

50 μl of 4 mg/ml PBI 4013 in DMSO was diluted to 1.25 ml with Nanopurewater, and 25 μl added to wells A1-D10 of plate A. 50n1 of 4 mg/ml PBI4424 was diluted to 1.25 ml with Nanopure water, and 25 μl added toE1-H10 of plate A. 50 μl of 4 mg/ml PBI 3048 was diluted to 1.25 ml withNanopure water, and 25 μl added to A1-D10 of plate B. 50 μl of 4 mg/mlPBI 4425 was diluted to 1.25 ml with Nanopure water, and 25 μl added toE1-H10 of plate B. 25 μl of Nanopure water was added to rows A and E ofboth plates. 17 μl of Nanopure water was added to rows B and F of bothplates; and 8 μl of Nanopure water was added to rows C and G of bothplates

A hydrogen peroxide solution (30% hydrogen peroxide, Sigma H1009-5 ml)was diluted to 60 μM hydrogen peroxide with Nanopure water, and 25 ti ofthe 60 μM hydrogen peroxide was added to rows D and H of both plates;17n1 of 60 μM hydrogen peroxide was added to rows C and G of bothplates; and 8 μl of 60 μl hydrogen peroxide was added to rows B and F ofboth plates. Both plates A and B were then gently mixed for 70 minutes.

A bottle of Reconstitution Buffer with Esterase (Promega V144B) wasthawed and used to reconstitute a Luciferin Detection Reagent (PromegaV859B) to create a reconstituted Luciferin Detection Reagent (“LDR”). 15ml of 1M HEPES buffer, pH 7.5 was mixed with 25 ml of the LDR, and 80n1added to wells A1-H10 of two new luminometer plates (“R1” and “R2”).

Following incubation, 20 μl of the contents of wells A1-H10 of plate Awas transferred to the same wells in plate R1, and 20 ti of A1-H10 ofplate B was transferred to the same wells in plate R2. Plate R1 and R2were then incubated at room temperature for 15 minutes, andbioluminescence was measured with a GloMax® luminometer (Promega Corp).

Bioluminescence from duplicate wells was averaged, and the averagerelative light units (RLUs) from samples containing peroxide werecompared to those from the samples not containing peroxide incubated atthe same pH (Table 1).

TABLE 1 pH 6.87 pH 7.41 pH 7.73 pH 8.04 pH 9.18 H₂O₂ (μM) PBI 0 421,702469,602 573,023 585,008 700,831 4013 5 580,978 760,900 1,089,7171,534,619 3,678,815 10 680,475 1,020,315 1,634,356 2,485,677 5,433,57715 793,789 1,282,281 2,209,227 3,373,538 7,062,631 PBI 0 17,522 16,18117,087 18,602 17,794 4424 5 18,952 17,583 18,681 20,935 22,309 10 19,89117,508 19,298 20,554 20,333 15 20,263 19,332 25,729 21,486 20,453 H₂O₂(uM) PBI 0 392,535 418,478 470,830 561,030 819,156 3048 5 521,375794,181 1,200,842 1,831,013 3,834,248 10 717,216 1,302,514 2,057,7803,137,287 6,426,397 15 833,785 1,709,044 2,713,251 4,193,451 7,447,757PBI 0 10,464 10,266 9,302 10,312 9,428 4425 5 13,296 14,549 13,73915,130 19,728 10 14,491 14,768 17,086 19,258 26,859 15 14,105 14,72515,789 18,905 24,236

As can be seen in Table 1, the light emitted from two of the compounds,PBI 4013 and PBI 3048, increased strongly upon addition of hydrogenperoxide at all pH values. However, greater light signal increases wereseen as the pH increased from pH 6.87 to pH 9.18. The light signalincreases measured for the other two compounds, PBI 4425 and PBI 4424,were much smaller upon hydrogen peroxide addition than was seen for PBI3048 or PBI 4013.

These results indicate that luciferin borates can be used to detect andmeasure hydrogen peroxide at low levels, but that small structuralchanges in the particular compounds can have very dramatic effects onthe increase of the relative strength of the light signal. In addition,these results indicate that a stronger light signal is obtained fromreactions of luciferin borates with hydrogen peroxide at pH values abovepH 8.0.

Example 15 Comparison of Light Generation from Compounds of the PresentInvention Following Incubation with Hydrogen Peroxide

In this experiment, various compounds were tested for their ability todetect hydrogen peroxide in different pH solutions. Several compoundsare shown to give much better signal strength and signal to backgroundratios than PBI 3048 (Example 23).

4 mg/ml solutions of compounds PBI 4480, 4481, 4452, 4470, 4472, 3048(FIGS. 1 and 2) were made by dissolving them in DMSO (Fluka 41641). 30μl of the compound solutions was diluted to 1.5 ml with Nanopure water,and 50 μl aliquots added as follows into a white, 96-well luminometerplate: PBI 4481, row A1-12; PBI 4480, row B1-12; PBI 4452, row C₁₋₁₂;PBI 3048, row D1-12; PBI 4472, row E1-12 and PBI 4470, row F1-12.

25 μl of 200 mM TRIS buffer, pH 7.6 was added to wells A1-F4, 25 μl of200 mM TRIS buffer, pH 8.8 was added to wells A5-F8, and 25 μl of 200 mMTris buffer, pH 10.4 was added A9-F 10.

30% hydrogen peroxide (Sigma H 1009-5 ml) was diluted to 40 μl inNanopure water, and 25 μl added to columns 3, 4, 7, 8, 11 and 12 of theplate. 25 μl water was added to columns 1, 2, 5, 6, 9, and 10 of theplate, and the plate incubated at room temperature for 60 minutes.

A conversion solution was made by mixing 100 μl of 100 mM D-cysteine, 1ml Tris Buffer pH 8.8 and 8.9 ml nanopure water. After mixing, 80 μl ofthe solution was added to wells A1-F12 of a new luminometer plate.

After incubation, 20 μl of sample from wells A1-F 12 of the first platewas transferred to the same well in the new luminometer plate. The newplate was incubated at room temperature for 5 minutes, 100 μl of LDR(Example 23) was added to wells A1-F12, and bioluminescence detected ona Glo Max® luminometer (Promega).

The RLUs for the duplicate wells were averaged. The averages for thewells containing hydrogen peroxide were compared to the wells withouthydrogen peroxide at the same pH (Table 2). BZT is used to designatebenzothiazole derivatives of the compounds.

TABLE 2 pH 7.6 pH 8.8 pH 10.4 No With With No H₂O₂ H₂O₂ No H₂O₂ H₂O₂H₂O₂ With H₂O₂ BZT PBI 4452 607,036 1,182,338 632,327 2,790,924 921,93514,577,312 Borates PBI 4470 492,941 701,300 468,729 1,525,058 819,41613,391,102 BZT Borate PBI 4472 102,925 1,854,434 163,710 5,175,361337,921 8,159,216 Esters PBI 4481 124,246 873,626 135,530 2,854,279230,459 7,474,603 PBI 4480 146,664 657,132 179,821 1,563,422 265,3155,295,463 Luciferin PBI 3048 569,141 1,726,143 558,869 2,499,869 558,3333,768,437 Borate

The Signal to Background (S/B) ratio is used to compare assay reagentswith very difference signal strengths. An assay with a higher S/B ratiois often considered a better assay than one with a smaller S/B ratio(with all other factors being relatively equal). The Signal (withperoxide) to Background (no peroxide) values for this Example are shownin Table 3.

TABLE 3 Signal to Background Ratios pH 7.6 pH 8.8 pH 10.4 BZT BoratesPBI 1.9 4.4 15.8 4452 PBI 1.4 3.3 16.3 4470 BZT Borate PBI 18.0 31.624.1 Esters 4472 PBI 7.0 21.1 32.4 4481 PBI 4.5 8.7 20.0 4480 LuciferinBorate PBI 3.0 4.5 6.7 3048

Even though PBI 4452 and 4470 are borates, they both produce greatersignals with peroxide at pH 10.4 and have a greater S/B ratio at that pHthan PBI 3048. This property of the compounds can make them more usefulin measuring hydrogen peroxide generated in vitro in enzymaticreactions. On the other hand, the borate esters (PBI 4472, 4481 and4480) give higher S/B ratios at lower pH than either class of boratessuggesting that they may perform better in experiments using livemammalian cells in culture where cells cannot be grown at a pH of pH10.4. In addition, although they generate a somewhat lower total signalwhen compared to the BZT borates under some conditions, the borateesters generate an excellent signal to background that could be used ifsignal strength is not minimal.

Example 16 Detection of Reactive Oxygen Generation Using LuciferinBorates

In this example, various compounds shown to react with hydrogen peroxidewere incubated with cells treated with menadione and 4-aminobiphenyl,two compounds that can generate reactive oxygen species (ROS). Menadionecauses the direct production of reactive oxygen within the cell and is avery strong inducer of a ROS effect (Sun, J S et. al. Cell Mol Life Sci(1997) vol 53 pp 967-76). 4-aminobiphenyl is believed to requiremetabolic conversion to a quinine-like structure before being able toinduce the formation of a ROS Makena, P and Chung, K T. Environ. Mol.Mutagen. (2007), vol 48, pp 404-413). One of the borate compounds, PBI4458, has been shown to give a much stronger signal under conditionsthat generate reactive oxygen within the cell. This exampledemonstrates: 1) a method to detect the formation of reactive oxygenspecies in cells upon application of various treatments using thecompounds of the present invention, and 2) the method of 1) can be usedto detect reactive species in both attached (e.g., HepG2) and suspension(e.g., Jurkat) cell lines.

A 40 mM solution of 4-aminobiphenyl (Sigma A2898-1 g) was made bydissolving 2.5 mg in 375 μl of DMSO (Fluka) to create a 40 mM solution.50 μl of 4 mg/ml PBI 4458 in DMSO was diluted to 1 ml with HBSS buffer(Invitrogen), and 100 μl added to wells A1 and A2 of a microtiter plate.A 10 μl sample of 40 mM 4-aminobiphenyl was mixed with 190 μl of theremaining PBI 4458 in HBSS, and 60 μl added to wells B1 and B2 and 30 μlto wells C1 and C2. A 10 μl sample of 40 mM menadione in DMSO was mixedwith 190 μl of the remaining PBI 4458 in HBSS, and 60 μl added to wellsD1 and D2 and 30 μl to E1 and E2. The volume in wells B1 to E2 was thenadjusted to 100 μl by addition of remaining PBI 4458 in HBSS.

50 μl of 4 mg/ml PBI 4480 in DMSO was diluted to 1 ml with HBSS buffer,and 100 μl placed in wells A3 and A4 of the microtiter plate. A 10 μlsample of 40 mM 4 aminobiphenyl was mixed with 190 μl of the remainingPBI 4480 in HBSS, and 60 μl added to wells B3 and B4 and 30 μl to wellsC3 and C4. A 10 μl sample of 40 mM menadione in DMSO was mixed with 190μl of the remaining PBI 4480 in HBSS, and 60 μl added to wells D3 and D4and 30 μl added to wells E3 and E4. The volume in wells B3 to E4 wasthen adjusted to 100 μl by addition of left over PBI 4480 in HBSS.

50 μl of 4 mg/ml PBI 3048 in DMSO was diluted to 1 ml with HBSS, and 100μl added to wells A5 and A6 of the microtiter plate. A 10 μl sample of40 mM 4 aminobiphenyl was mixed with 190 μl of the remaining PBI 3048 inHBSS, and 60 μl added to wells B5 and B6 and 30 μl added to wells C5 andC6. A 10 μl sample of 40 mM menadione in DMSO was mixed with 190 μl ofthe remaining PBI 3048 in HBSS, and 60 μl added to wells D5 and D6 and30 μl added to wells E5 and E6. The volume in wells B5 to E6 was thenadjusted to 100 μl by addition of the remaining PBI 3048 in HBSS.

Two cell culture plates were seeded with cells resuspended in HBSS. Oneplate contained 20,000 Jurkat cells/well in wells A1-H6. The other platecontained 20,000 HepG2 cells/well in wells A1-H6. Both plates wereincubated overnight in DMEM with 10% fetal bovine serum (FBS). The mediafrom the HepG2 cells was removed, discarded, and replaced with 50 μl ofHBSS. A 50 μl sample of wells A1-H6 from the plate with the various PBIcompounds was transferred to the corresponding wells in the two cellculture plates, and incubated for 60 minutes in a 37° C., 5% CO₂incubator.

50 μl of LDR (Example 23) and 25 μl of 1 mM D-cysteine in 100 mM HEPESbuffer, pH 7.5 was added to all wells of a new luminometer plate. Afterincubation, a 25 μl sample of wells A1-H6 containing Jurkat cells wasadded to wells A1-H6 of the new luminometer plate, and a 25 μl sample ofwells A1-H6 containing HepG2 cells was added to wells A7-H12 of the newluminometer plate. The plate was incubated at room temperature for 15minutes, and bioluminescence detected on a GloMax® luminometer.

The bioluminescence from the duplicate samples was averaged and comparedto the values of the cells in A row (Table 4).

TABLE 4 Jurkat HepG2 PBI PBI PBI PBI PBI PBI 4458 4480 3048 4458 44803048 No Effector 174,206 34,646 413,753 117,111 19,627 407,917 600 μM4ABP 195,096 29,277 366,188 169,952 21,023 383,471 300 μM 4ABP 213,05929,810 363,182 163,031 20,574 364,353 600 μM Menadione 895,269 27,535403,013 728,404 20,509 408,177 300 μM Menadione 742,717 29,806 434,344622,230 20,788 426,802

The values for the different levels of 4-aminobiphenyl and menadionewith PBI 4458 are above those for the cells that were not given anyeffector, as was expected. Also, the magnitude of the response formenadione was much greater than for 4-aminobiphenyl. The values for PBI4480 and PBI 3048 do not show as clear of a response to the menadione or4 aminobiphenyl. The reduced response is not surprising since thesecompounds showed a less then strong response to direct hydrogen peroxideaddition as seen in Example 23.

These results indicate that PBI 4458 allows sensitive detection of anagent that generates reactive oxygen in mammalian cells. In addition,the results clearly demonstrate that not all borate analogs attached toa benzothiazole or luciferin will generate signals when used with cells.In fact, some of the compounds generate signals with cells in bufferthat may be too weak for reliable detection of the effects of compoundson reactive oxygen generation in mammalian cells in culture.

Example 17 Buffer Effects on Luciferin Generation

In this example, borate compounds of the present invention wereincubated in various buffers to demonstrate that improved signalstrength can be obtained using the proper buffer. In addition, theexample demostrates that signal generation in two buffers, DMEM and HBSSbuffer, worked well with mammalian cells. Some compounds generatedgreatly stronger signals in DMEM than in HBSS even though both areformulated to have the same pH value thus demonstrating that theparticular compounds of the present invention may be very advantageousto use with mammalian cells in a media such as DMEM.

A. Demonstration of Signal Strength Differences Due to Reaction pH andBuffer Composition.

A 30 μl sample of 4 mg/ml PBI 4458 in DMSO was diluted to 1.5 ml withNanopure water, and 50 μl added to wells A1-12 and E1-12 of a microtiterplate. A 30 μl sample of 4 mg/ml PBI 4472 in DMSO was diluted to 1.5 mlwith Nanopure, and 50 μl added to wells B1-12 and F1-12 of the plate. A30 μl sample of 4 mg/ml PBI 4480 in DMSO was diluted to 1.5 ml withNanopure water, and 50 μl added to wells C₁₋₁₂ and G1-12 of the plate. A30 μl sample of 4 mg/ml PBI 4481 in DMSO was diluted to 1.5 ml withNanopure water, and 50 μl added to wells D1-12 and H1-12 of the plate.

To the plate, a 50 μl sample of 200 mM KHPO₄, pH 7.4 was added to wellsA1-D4; 50 μl 200 mM Tris buffer, pH 7.4 was added to wells A5-D8; 50 μl250 mM HEPES buffer, pH 7.5 was added to wells A9-D12; 50 μl 200 mMKHPO₄ buffer, pH 9.2 was added to wells E1-H4; 50 μl 250 mM Tris buffer,pH 10.4 was added to wells E5-H8; and 50 μl 100 mM CAPS buffer, pH 10.4was added to wells E9-H12.

A sample of hydrogen peroxide (30%, Sigma, H1009-5 ml) was diluted to 10μM with Nanopure water, and 100 μl added to columns 3, 4, 7, 8, 11, 12wells A-H of the plate. 100 μl Nanopure water was added to columns 1, 2,5, 6, 9 and 10 of the plate. The plate was incubated at room temperaturefor 15 minutes.

A conversion solution was made by mixing 5 ml of 1M HEPES buffer, pH7.5, 4.9 ml of Nanopure water and 100 μl of 100 mM D-cysteine (inNanopure water), and 25 μl added to all wells of a luminometer plate.

After incubation, 25 μl of sample from all wells in the plate wastransferred and mixed into the corresponding wells in the luminometerplate, incubated 2-3 minutes at room temperature, and 50 μl LDR (Example23) added. The luminometer plate was incubated for 15 minutes at roomtemperature, and bioluminescence detected on a GloMax® luminometer.

Additional samples were taken at 60 and 105 minutes and added to a freshluminometer plates containing 25 μl of conversion solution in all wells.The plates were then incubated for 15 minutes at room temperature, andbioluminescence detected as previously described.

The RLUs from duplicate wells were averaged, and the values from thesamples containing hydrogen peroxide were compared to the correspondingaverage of the samples that did not contain hydrogen peroxide (Tables 5,7 and 9), and the signal to background determined (Tables 6, 8 and 10).

TABLE 5 Net Average Signals at 15 Minutes KPO₄ Tris KPO₄ CAPS Ph 7.4 pH7.4 HEPES pH 7.5 pH 9.2 TRIS pH 10.4 pH 10.4 PBI 148,190 150,205 117,752937,111 2,348,900 4,577,774 4458 PBI 43,867 121,480 58,399 400,0871,530,303 3,154,440 4472 PBI 3,538 7,845 −234 37,330 743,427 1,799,0154480 PBI 16,032 29,382 15,697 189,573 1,467,615 3,139,869 4481

TABLE 6 Signal to Background Ratios at 15 Minutes KPO₄ Tris HEPES KPO₄TRIS CAPS pH 7.4 pH 7.4 pH 7.5 pH 9.2 pH 10.4 pH 10.4 PBI 2.2 2.2 2.06.4 15.2 13.2 4458 PBI 3.1 6.6 3.9 11.0 32.9 17.0 4472 PBI 1.1 1.2 1.01.7 11.1 12.1 4480 PBI 1.5 1.9 1.4 4.7 23.2 15.0 4481

TABLE 7 Net Average Signals at 60 Minutes KPO₄ Tris KPO₄ CAPS pH 7.4 pH7.4 HEPES pH 7.5 pH 9.2 TRIS pH 10.4 pH 10.4 PBI 615,057 626,927 494,9582,490,775 3,795,644 5,831,485 4458 PBI 102,614 371,397 159,982 781,2963,777,236 5,425,701 4472 PBI 38,198 61,031 29,727 218,078 1,903,8854,256,769 4480 PBI 48,355 111,440 56,121 494,981 2,780,433 4,514,9984481

TABLE 8 Signal to Background Ratios at 60 Minutes KPO₄ Tris HEPES KPO₄TRIS CAPS pH 7.4 pH 7.4 pH 7.5 pH 9.2 pH 10.4 pH 10.4 PBI 4.5 4.6 4.59.5 19.4 12.1 4458 PBI 4.9 13.2 8.0 14.7 34.2 15.4 4472 PBI 1.6 2.0 1.73.6 17.9 14.7 4480 PBI 2.2 3.5 2.5 8.3 27.8 15.6 4481

TABLE 9 Net Average Signals at 105 Minutes KPO₄ Tris KPO₄ CAPS pH 7.4 pH7.4 HEPES pH 7.5 pH 9.2 TRIS pH 10.4 pH 10.4 PBI 1,297,857 1,274,0611,033,511 4,925,484 5,482,012 8,063,108 4458 PBI 168,787 658,967 259,7251,380,626 6,279,007 7,554,957 4472 PBI 86,881 162,663 75,066 587,8673,541,742 7,150,225 4480 PBI 108,403 245,489 111,757 974,784 4,441,9566,646,007 4481

TABLE 10 Signal to Background Ratios at 105 Minutes KPO₄ Tris HEPES KPO₄TRIS CAPS pH 7.4 pH 7.4 pH 7.5 pH 9.2 pH 10.4 pH 10.4 PBI 5.4 6.0 5.79.1 15.7 9.6 4458 PBI 5.3 13.6 8.2 14.6 26.5 11.3 4472 PBI 1.9 2.6 2.35.1 17.5 12.6 4480 PBI 2.7 4.4 3.0 7.7 24.2 12.3 4481

As seen previously, the bioluminescence signal strength is greater athigher pH values, but there is variation in the signal strength at aparticular pH value dependent upon the buffer. For example, even thoughthe buffer pH is nearly identical for the reactions performed using thebuffers KPO₄, pH 7.4, Tris, pH 7.4 and HEPES, pH 7.5, the net signalsseen for PBI 4472 at 105 minutes are different resulting in differentsignal to background values (5.3, 13.6 and 8.2 for PBI 4472 at 105minutes in KPO₄, Tris pH 7.4, and HEPES pH 7.5, respectively).

B. Signal Differences in Mammalian Cell Culture Media Versus aPhysiological Buffer

A 100 μl sample of 4 mg/ml DMSO solution of PBI 4458 was diluted to 500μl with HBSS buffer (Invitrogen 14025-092), and 50 μl added to wellsA1-A9 of a fresh microtiter plate (“DISP”). 100 μl 4 mg/ml DMSO solutionof PBI 4472 was diluted to 500 μl with HBSS, and 50 μl added to wellsE1-E9. 90 μl HBSS was added to wells A1-B9 of two new microtiter plateslabeled “RT” and “37C”. 90 μl DMEM was added to wells C1-D9 of bothplates.

A sample of 30% hydrogen peroxide (Sigma H1009-5 ml) was diluted to 1 mMwith Nanopure water, and 7.5 μl 1 mM hydrogen peroxide diluted to 1.5 mlwith HBSS, and 90 μl added to wells A4-B6 of plates RT and 37° C. 15 μl1 mM hydrogen peroxide was diluted to 1 mM with HBSS, and 90 μl added towells A7-B9 of both plates. 90 μl of HBSS was added to wells A1-B3, and90 μl of DMEM added to wells C1-D3 of both plates. 7.5 μl 1 mM hydrogenperoxide was diluted to 1.5 ml with DMEM, and 90 μl added to wells C4-D6of both plates. 15 μl 1 mM hydrogen peroxide was diluted to 1.5 ml withDMEM and 90 μl added to wells C7-D9 of both plates.

10 μl samples of row A of plate “DISP” was transferred to rows A and Cof plates RT and 37° C., and 10 μl of row B of plate “DISP” transferredto rows B and D of plates RT and 37° C. The 37° C. plate was incubatedat 37° C., 5% CO₂, with plate RT incubated at room temperature. Bothplates were incubated for 30 minutes.

A conversion solution was made by mixing 7.5 ml of 1M HEPES, pH 8.0, 7.4ml Nanopure water and 100 μl 100 mM D cysteine. After mixing, 75 μl wasadded to all wells of a new luminometer plate.

After incubation, 25 μl from wells A1-D9 of the 37° C. plate wastransferred to wells E1-H9 of the new luminometer plate, 25 μl of wellsA1-D9 of the RT plate was transferred to wells A1-D9 of new luminometerplate, and the plate incubated at room temperature for 2-3 minutes. A100 μl sample of LDR (Example 23) was added to wells A1-H9, incubatedfor 15 minutes at room temperature, and bioluminescence detected on aGloMax® luminometer. After reading, the duplicate wells were averaged,and the averaged values with peroxide addition were compared to thosewithout peroxide (Table 11).

TABLE 11 Averaged Relative Light Unit Values RT plate 37 C plate MediaCompound 0 uM Per 5 uM Per 10 uM Per 0 uM Per 5 uM Per 10 uM Per HBSSPBI 4458 853,268 6,111,214 10,641,411 434,728 2,791,805 4,912,905 HBSSPBI 4472 103,738 1,106,195 2,058,421 44,007 424,851 715,803 DMEM PBI4458 2,075,191 8,556,256 16,016,143 986,772 5,686,033 12,130,782 DMEMPBI 4472 921,496 4,304,645 8,071,553 400,077 3,083,589 5,340,569

As seen in Table 11, the “0 μM peroxide” values at 37° C. were lowerthan at RT, and signals from reactions in DMEM were greater than inHBSS, suggesting that some component in DMEM (either on not present orat a different concentration in HBSS) affects the signal strength. Whilethe exact cause of the signal differences seen between reactionsperformed at room temperature vs. 37° C. is not known, the backgroundreduction seen upon incubating these two compounds in DMEM at 37° C.suggests they will work well as hydrogen peroxide sensors with mammaliancells grown in DMEM. The strong signal from PBI 4458 in media comparedto PBI 3048 or 4480 in B may be the result of this background reduction.Additional examples for 9547 US00

Example 18 Detection of Reactive Oxygen Generation Using CoelenterazineBorates

PBI 4759 (10 mM; final concentration 12.5 uM), various concentrations ofhydrogen peroxide and 40 ng/ml NanoLuc™ luciferase enzyme (PromegaCorporation) were added to wells of a 96-well assay plate containing 200mM Tris—Cl either at pH 8.5 or pH 9.0. Luminescence was detected on aGloMax® luminometer.

The results indicate the PBI 4759 allows sensitive detection of hydrogenperoxide (FIG. 3). The signal is proportional to the level of hydrogenperoxide (H2O2) present in the reaction and is also dependent upon thepresence of NanoLuc™ luciferase enzyme (NL). Note that although thefirst boxed set of numbers in FIG. 3 are labeled with columns with “NLwith H2O2” and “No NL with H2O2”; in fact these reactions did notcontain any H₂O₂ and acted as a control for the other additions.

Example 19 Background Reduction of PBI 4472

PBI 4472 (50 μM) was incubated with assay buffer (100 mM Tris base, pH10.4) for 15 minutes at room temperature. The samples were then eithertreated with a background reduction reagent (BRR; TCEP) or remaineduntreated. After a 15 minute incubation at room temperature, luciferindetection reagent with 1 mM D-cysteine (Promega Corporation) was addedto the samples and incubated for 20 minutes at room temperature.Luminescence was detected on a Tecan F500 plate reader.

The results demonstrate that the addition of a reduction reagent canreduce background that may be seen with the luciferin borates such asPBI 4472 (FIG. 4).

All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification this inventionhas been described in relation to certain preferred embodiments thereof,and many details have been set forth for purposes of illustration, itwill be apparent to those skilled in the art that the invention issusceptible to additional embodiments and that certain of the detailsdescribed herein may be varied considerably without departing from thebasic principles of the invention.

1. A compound according to Formula (II):

wherein R₁₁ is boronic acid or a borate ester; R² is —(CH₂)_(n)-T orC₁₋₅ alkyl; R⁶ is selected from the group consisting of —H, —OH,—NH₂—OC(O)R or —OCH₂OC(O)R; R⁸ is selected from the group consisting of

H or lower cycloalkyl; wherein R³ and R⁴ are both H or both C₁₋₂ alkyl;n is 0 to 3; each R is independently a C₁₋₂ alkyl; T is aryl,heteroaryl, substituted aryl, substituted heteroaryl or cycloalkyl; L₂is a linker; and the dashed bonds indicate the presence of an optionalring which may be saturated or unsaturated.


2. A compound according to claim 1, wherein L₂ is A is —C₆(R₁₀)₄- or—(CR₂₁═CR₂₁)_(n)— or —O—C₆(R₁₀)₄- or —S—C₆(R₁₀)₄- or —NR′ —C₆(R₁₀)₄- ora direct bond; R′ is H or C₁₋₄ alkyl; each R₃ is independently halo, H,C₁₋₄ alkyl, C₁₋₄ hydroxyalkyl, or C₁₋₄ alkylcarboxylic acid; each R₁₀ isindependently H, halo, CH₃, OCH₃, or NO₂; each R₂₁ is independently H orCH₃; n is 1 or 2; and X is selected from a direct bond, —C(O)—, and—C(O)NR₂₂, where R₂₂ is H or C₁₋₄ alkyl.
 3. A compound according toclaim 1, wherein R₁₁ is

and each R₁₂ and R₁₃ is independently selected from H, C₁₋₄ alkyl, CF₃,phenyl or substituted phenyl; or R₁₂ and R₁₃ together can be an alkylring having from 3-7 carbons or can be replaced by a fused 6-memberedaromatic ring.
 4. A compound according to claim 1, wherein R₁₁ is

and each R₁₄, R₁₅ and R₁₆ is independently selected from H, C₁₋₄ alkyl,CF₃, phenyl and substituted phenyl; or both R₁₅ together can form analkyl ring having from 3-7 carbons; R₁₄ and R₁₅ together or R₁₅ and R₁₆together can be an alkyl ring having from 3-7 carbon atoms or can bereplaced by a 6-membered aromatic ring.
 5. A compound according to claim1, wherein: R² is

and X is O or S.
 6. A compound according to claim 1, wherein R² is C₂₋₅straight-chain alkyl.
 7. A compound according to claim 1, wherein R⁸ is

lower cycloalkyl or H, wherein R³ and R⁴ are both H or C₁₋₂ alkyl.
 8. Acompound according to claim 1, wherein, R⁸ is benzyl.
 9. A method ofdetecting hydrogen peroxide in a cell comprising: (a) contacting cellswith a compound according to claim 1; (b) adding a luciferase reactionmixture to the contacted cells; and (c) measuring bioluminescencethereby detecting the presence of hydrogen peroxide in the cell.
 10. Themethod of claim 9, wherein the luciferase reaction mixture comprisesD-cysteine.
 11. The method of claim 9, wherein the cell is in an animalor in a sample.
 12. A method of detecting hydrogen peroxide in a cellcomprising: (a) contacting cells with a compound according to claim 1 ina first reaction vessel to form an incubation mixture; (b) transferringat least a portion of the incubation mixture to a second reactionvessel; (c) adding a luciferase reaction mixture to the second reactionvessel; and (d) measuring bioluminescence thereby detecting the presenceof hydrogen peroxide in the cell.
 13. The method of claim 12, whereinthe luciferase reaction mixture comprises D-cysteine.
 14. The method ofclaim 12, wherein the cell is in an animal or in a sample.
 15. A methodof detecting hydrogen peroxide in a sample comprising: (a) contacting asample with a compound according to claim 1 to form a first mixture; b)contacting at least a portion of the first mixture with a luciferasereaction mixture and D-cysteine and c) measuring bioluminescence therebydetecting the presence of hydrogen peroxide in the sample.
 16. Themethod of claim 15, wherein the D-cysteine is part of the reactionmixture for a luciferase reaction mixture.
 17. The method of claim 15,wherein the sample comprises a cell, cell medium or physiological samplefrom an animal.
 18. A method of determining the effect of a testcompound or test condition on the presence or amount of hydrogenperoxide in a sample comprising: a) treating a sample with a testcompound or test condition, b) contacting the sample from a) with acompound according to claim 1; c) adding a luciferase reaction mixtureto the sample; d) measuring bioluminescence.
 19. The method of claim 18,wherein the sample comprises a cell, cell medium or physiological samplefrom an animal.
 20. A kit comprising a compound according to claim 1.21. A compound of formula